不同氧浓度对兔耳软骨细胞增殖、凋亡、迁移功能的影响

Effects of oxygen concentration on proliferation, apoptosis, and migration ability of in vitro cultured rabbit auricle chondrocytes

目的:探讨不同氧浓度在不同培养时间对兔耳软骨细胞增殖、凋亡和迁移功能的影响。方法:日本大耳白兔6只,取其双侧耳廓软骨,进行细胞培养,以第2代软骨细胞进行实验。实验分为5组:A组于常氧(氧浓度为21%)条件下培养软骨细胞(对照组),B组于5%氧浓度下培养软骨细胞12 h,C组于5%氧浓度下培养36 h,D组于1%氧浓度下培养12 h,E组于1%氧浓度下培养36 h。培养完毕后进行观察,CCK-8法检测各组细胞增殖能力(吸光度A值),绘制细胞增殖曲线;Annexin V-FITC细胞凋亡检测试剂盒联合流式细胞仪检测细胞凋亡率;细胞划痕实验结合相对面积法检测各组细胞迁移能力。多组间比较采用单因素方差分析,2组间比较采用LSD-t法,以P<0.05为差异有统计学意义。结果:在细胞增殖方面,培养至第6天,A、B、C、D、E组吸光度A值分别为1.38±0.29、1.59±0.27、1.37±0.22、2.06±0.64、2.23±0.56,5组之间比较,差异有统计学意义(F=4.207,P=0.012),其中D组和A组之间差异有统计学意义(t=-2.487,P=0.022),E组和A组之间差异有统计学意义(t=-3.095,P=0.006)。在第7天,5组的吸光度A值依次为1.72±0.30、2.26±0.44、2.30±0.29、2.49±0.73、2.74±0.54,5组间比较,差异有统计学意义(F=2.948,P=0.046),D组和A组(t=-2.480、P=0.022)、E组和A组(t=-3.287、P=0.004)之间,差异均有统计学意义。在细胞凋亡方面,A、B、C、D、E组细胞凋亡率依次为(2.97±1.14)%、(3.92±1.14)%、(3.38±0.83)%、(1.54±0.50)%、(0.99±0.59)%。5组间比较,差异有统计学意义(F=7.957,P=0.001),其中D组与A组(t=-2.300、P=0.036)、E组与A组(t=-3.180、P=0.006)、B组与D组(t=3.812、P=0.002)、C组与E组(t=3.832、P=0.002)之间,差异均有统计学意义。在细胞迁移方面,划痕后12 h,A、B、C、D、E组细胞迁移率依次为(13.10±3.32)%、(9.23±3.56)%、(10.60±2.03)%、(13.33±1.72)%、(15.32±4.72)%。5组间比较,差异有统计学意义(F=3.278,P=0.027),其中D组与B组(t=2.183、P=0.039)、C组与E组比较(t=-2.513、P=0.019),差异有统计学意义;划痕后24 h,5组细胞迁移率依次为(19.7±2.97)%、(16.62±3.30)%、(18.99±2.61)%、(20.92±5.18)%、(25.29±5.83)%,5组间比较,差异有统计学意义(F=3.513、P=0.021),其中E组与A组(t=2.315、P=0.029)、E组与C组(t=2.609,P=0.015)比较,差异均有统计学意义。结论:当培养时间超过12 h时,与氧浓度为5%和21%时相比,1%氧浓度可使兔耳软骨细胞增殖能力更强、凋亡率更低、迁移率更高,且氧浓度高低对兔耳软骨细胞生物学特性的影响大于干预时间长短的影响。

Objective To investigate the effect of oxygen concentration on proliferation,apoptosis and migration of rabbit auricular chondrocytes at different time points of culture.Methods Bilateral auricular cartilage from 6 Japanese white rabbits were harvested and cultured,auricular chondrocytes at passage 2(P2)were used in this study.Five groups were set:Group A(control group):chondrocytes were cultured in atmospheric(21%)oxygen condition,group B:chondrocytes were cultured in 5%oxygen condition for 12 hours(h),group C:chondrocytes were cultured in 5%oxygen condition for 36 h,group D:chondrocytes were cultured in 1%oxygen condition for 12 h,and group E:chondrocytes were cultured in 1%oxygen condition for 36 h.Cell counting kit-8(CCK-8)assay was adopted to evaluate the proliferation of chondrocytes.Proliferation curves were drawn according to the absorbance value detected.Cell apoptosis was investigated by annexin V-FITC and flow cytometry.Cell scratch test was used to observe the migration ability.One-way ANOVA and LSD-t were used to verify differences between the groups.P<0.05 was considered as statistically significant.Results In terms of cell proliferation,on the sixth day of culture,the absorbance values of group A,B,C,D and E were 1.38±0.29,1.59±0.27,1.37±0.22,2.06±0.64,2.23±0.56 respectively.Significant difference was found between the five groups(F=4.207,P=0.012),and there were significant differences between group D and A(t=-2.487,P=0.022),group E and A(t=-3.095,P=0.006).On the seventh day of culture,the absorbance values of the five groups were 1.72±0.30,2.26±0.44,2.30±0.29,2.49±0.73,2.74±0.54.Significant difference was found between five groups(F=2.948,P=0.046),and there were significant differences between group D and A(t=-2.480,P=0.022),group E and A(t=-3.287,P=0.004).The apoptosis rate of group A,B,C,D and E were(2.97±1.14)%,(3.92±1.14)%,(3.38±0.83)%,(1.54±0.50)%,(0.99±0.59)%.The difference between groups for cell apoptosis were also significant(F=7.957,P=0.001),among which there were significant differences between group D and A(t=-2.300,P=0.036),group E and A(t=-3.180,P=0.006),group B and D(t=3.812,P=0.002),group C and E(t=3.832,P=0.002).As for cell migration,at the 12th hour,the cell migration rates of groups A,B,C,D and E were(13.10±3.32)%,(9.23±3.56)%,(10.60±2.03)%,(13.33±1.72)%,(15.32±4.72)%.Significant difference was found between the five groups(F=3.278,P=0.027).And there were significant differences between group D and B(t=2.183,P=0.039),group C and E(t=-2.513,P=0.019).At 24 h,the cell migration rates of the five groups were(19.7±2.97)%,(16.62±3.30)%,(18.99±2.61)%,(20.92±5.18)%,(25.29±5.83)%.Significant difference was found between five groups(F=3.513,P=0.021).And there were significant differences between group E and A(t=2.315,P=0.029),group E and C(t=2.609,P=0.015).Conclusions When cell culture time exceeded 12 h,1%oxygen condition induced higher proliferation,lower apoptosis,and higher migration ability than 5%or 21%oxygen conditions in cultured chondrocytes.Moreover,oxygen concentration had stronger influence on the biological characteristics of rabbit auricular chondrocytes compared to intervention time.